Protease activated receptor-2 (PAR2) is a unique G-protein coupled receptor (GPCR) in that it has no known endogenous extracellular ligand, but rather is activated by proteases including many serine proteases such as trypsin, tryptase, and cathepsin Q. Serine proteases cleave a section of the membrane bound receptor's extracellular N-terminus, exposing a new sequence which acts as a tethered ligand by binding to the receptor and initiating activation.
Distributed widely throughout the body, PAR2 has been implicated as a pro-inflammatory mediator in acute and chronic inflammatory diseases including arthritis, inflammatory bowel disease, pancreatitis, and cardiovascular diseases. PAR2 has also been reported as anti-inflammatory and protective in conditions such as gastric ulcer, colitis, asthma, and liver fibrosis, although this remains controversial. PAR2 activation has been linked to proliferation, metastasis and angiogenesis in many cancers including cancers or the stomach, colon, breast and pancreas. In this context, small-molecule modulators of PAR2 are of potential interest as a new class of anti-inflammatory, pro-inflammatory, anti-proliferative or proliferative agents.
Recently, interest has grown in inflammatory GPCRs as novel therapeutic targets for diet-induced obesity and metabolic syndrome after the identification of increased circulation of various pro-thrombotic molecules (such as plasminogen activator inhibitor-1, tissue factors) and other cysteine/serine proteases (such as cathepsins, caspases, tryptase, factor VII, factor X, trypsin-like serine proteases) that have been implicated in these disorders.
Human diets increasingly high in saturated fats and carbohydrates are thought to overload metabolic and immune systems, leading to obesity, metabolic dysfunction and impaired immunity. This excessive nutrient intake induces a chronic inflammatory state in adipose tissue promoting obesity, altering adipocyte function and immune cell distribution, both of which appear to trigger metabolic dysfunction. Chronic metabolic dysfunction can lead to obesity, type II diabetes and cardiovascular disease, their treatments representing major challenges to global health systems (Iyer, A., et al, Nat Rev Endocrinol 2009, 6, 71-82; Iyer, A. & Brown, L. Drug Discovery Today: Disease Mechanisms 2011). Modern sedentary lifestyles coupled with excessive caloric intake are important factors in initiating obesity and associated metabolic and cardiovascular disorders that are now collectively referred to as the ‘metabolic syndrome’ (Potenza, M. V. & Mechanick, J. I., Nutrition in Clinical Practice 2009, 24, 560-77; Simmons, R. K. et al. Diabetologia 2010, 53, 600-5).
Metabolic syndrome is associated with complications such as excessive visceral fat deposition, hypertension, impaired glucose and insulin homeostasis, insulin resistance, endothelial damage, cardiovascular hypertrophy, inflammation, vascular inflammation, atherosclerosis, ventricular contractile dysfunction, fibrosis and fatty liver disease. Identifying effective therapeutic and preventive options to treat this multi-factorial syndrome has proven to be very challenging, with an emerging focus on developing anti-inflammatory agents that can combat adiposity, metabolic and cardiovascular dysfunction as chronic inflammation has been shown to play a major role in both initiation and progression of obesity and metabolic syndrome.
Among the dynamic components of adipose tissue are adipocytes as well as many different immune cells such as macrophages, monocytes, T-cells and mast cells that contribute indirectly to adipocyte function. Alteration in the population of immune cells (especially macrophages) resident in adipose tissue early during the development of obesity is currently thought to propagate oxidative and inflammatory cascades triggering adiposity and metabolic dysfunction. Signals that initiate macrophage activation in adipose tissue are unknown. A growing body of evidence suggests that saturated fatty acids and lipid mediators produced locally by adipocytes and/or macrophages can, at least in part, participate in inflammatory cell activation, adipocyte growth, development and dysfunction and, therefore, contribute to obesity and metabolic disturbances. Various inflammatory lipid-induced G protein-coupled receptors (GPCRs) are thought to signal intracellular proteins that contribute to regulation of obesity, adipose tissue immune cell-dependent inflammation, insulin secretion and cardiovascular homeostasis.
Trypsin is a potent activator of PAR2 in the GI tract where pancreatic trypsin is found, and in colon, airway epithelium, neuronal and vascular endothelial cells, skin, intestine, kidney and pancreas where trypsinogen expression has been demonstrated. Mast cell tryptase is also an important activator of PAR2, being highly expressed in mast cells and strongly associated with many inflammatory, endocrine and other diseases. Pro-thrombotic factors such as tissue factor Vila have also been implicated in the cleavage of PAR2, exposing receptor activating sequences encoded within the N-terminus, thereby activating PAR2 signal transduction in adipose tissue and macrophages. Hexapeptides SLIGKV-NH2 and SLIGRL-NH2, corresponding to the tethered ligand human and murine sequences respectively, can activate human PAR2 in lieu of serine proteases, albeit at lower potency (μM instead of nM concentrations).
More potent peptide agonists have been created for PAR2. The hexapeptide analogue, 2-furoyl-LIGRL-NH2, has ˜20-fold higher agonist potency than SLIGRL-NH2 and is selective for PAR2 over PAR1 and PAR4. Other heterocyclic replacements for serine result in equipotent PAR2 agonists, while large aromatic groups in place of the C-terminal leucine impart a similar enhancement in PAR2 agonist potency (McGuire, J. J. et al, J Pharmacol Exp Ther 2004, 309, 1124-31; Barry G. D. et al, Bioorg Med Chem 2007, 27, 5552-7; Hollenberg, M. D., et al, J Pharmacol Exp Ther 2008, 326, 453-62; Boitano, C., et al, J Med Chem 2011, 54, 1308-13; Flynn, A. N., et al, J Biological Chem 2011, 286, 19076-88). Screening of 250,000 drug-like compounds produced two small molecule agonists of PAR2 with similar agonist potency to 2-furoyl-LIGRL-NH2, some selectivity for PAR2 and metabolic stability in vivo (Seitzberg, J. G., et al. J Med Chem 2008, 51, 5490-3).
The first known antagonist of PAR2 had affinity at only millimolar concentrations for the receptor and selectivity for PAR2 over non-PAR receptors is most unlikely (Kelso, E. B., et al. J Pharmacol ExpTher 2006, 316, 1017-24). A second antagonist reported for PAR2 is active at μM concentrations, but completely inactive against endogenous PAR2 activators like trypsin, tryptase and other proteases (Kanke, T, et al. Br J Pharmacol 2009, 158, 361-371) or has a dual function as an antagonist and agonist due to either partial agonist actions or possible agonist-directed signalling (Goh, F. G., et al. Br J Pharmacol 2009, 158, 1695-1704). More recently, a PAR2 antagonist with activity at low micromolar concentrations has been reported and was found to be selective for PAR2 over other PARs, reversibly inhibiting receptor activation by proteases and synthetic PAR2 agonists (Barry G. D. et al, J. Med. Chem. 2010, 53, 7428-40).
In one aspect the present invention advantageously provides a novel class of compounds that Can selectively modulate PAR2 when used at as low as micromolar or sub-micromolar concentrations. Depending upon structural characteristics, and intracellular pathways being examined, these novel compounds may act as either agonists or antagonists and be useful as tools for biological studies or as agents for anti-inflammatory, pro-inflammatory, anti-proliferative or proliferative therapies.
Current treatments for obesity act by reducing or controlling weight in patients by altering appetite, metabolism or absorption of calories and nutrients from food, for example, Orlistat (Xenical) is currently approved by the FDA for long term use. Orlistat reduces intestinal fat absorption by inhibiting pancreatic lipase. A second medicament, Rimonabant (Acomplia) works via a specific blockade of the endocannabinoid system. The FDA approved combination drug Qsymia comprises phentermine, a stimulant that suppresses the appetite and topiramate, an anticonvulsant. However, until now, the role of inhibitors of PAR2 in the reversal or attenuation of the symptoms of metabolic syndrome has not been reported.
Surprisingly, it has now been found that particular antagonists of PAR2 are able to reverse, prevent or attenuate obesity, metabolic syndrome and its associated diseases and disorders including adipose inflammation, type II diabetes, fibrosis and cardiovascular diseases.
Accordingly, in another aspect the present invention advantageously provides the use of PAR2 antagonists in the treatment and/or prevention of metabolic syndrome, obesity, adipose inflammation, type II diabetes, fibrosis and cardiovascular disease. Aspects of the present invention are based on the revelation that the action of PAR2 antagonism is effective in treatment and/or prevention of metabolic syndrome, obesity, insulin and glucose intolerance that are characteristic of type II diabetes, cardiovascular irregularities that are characteristic of cardiovascular diseases, and fibrosis as defined by collagen deposition.
Crohn's disease and ulcerative colitis are common forms of inflammatory bowel disease (IBD) that share common pathologies. Ulcerative colitis affects the colon and rectum and Crohn's disease affects multiple regions of the colon and ileum, and each condition has characteristic pasterns of ulcerative mucosa. Surprisingly, it has now been found that antagonists of PAR2 are able to prevent or attenuate inflammatory bowel disease with improved efficacy compared to current treatments. Accordingly, in another aspect the present invention advantageously provides the use of PAR2 antagonists in the treatment and/or prevention of inflammatory bowel disease.